Process for the extraction and purification of proteins from culture media producing them

ABSTRACT

The process is applicable to the supernatant of engineered yeast cells disrupted in the presence of a non-ionic detergent; it comprises the precipitation of contaminants by polyethylene glycol and the treatment of this latter supernatant with either a bivalent metal cation or, after eventual ultrafiltration, with ammonium sulfate.

This is a divisional of application Ser. No. 719,601 filed Apr. 3, 1985,now U.S. Pat. No. 4,683,294.

FIELD OF THE INVENTION

The invention relates to an improved process for the extraction andpurification of proteins from culture media producing them; moreparticularly, the process relates to the extraction and purification ofhepatitis B virus surface antigen and alpha-1-antitrypsin produced byrecombinant DNA technique in engineered cell cultures, more particularlyengineered yeast strains cultures.

BACKGROUND OF THE INVENTION

Different microorganisms, among which bacteria and yeasts can be used ashost organisms for different plasmids containing a DNA moleculecomprising a nucleotide sequence coding for a specific protein. Amongthese microorganisms, yeasts are presently preferred and currently used.For example, European Patent application publication No. 0 106 828discloses the use of Saccharomyces cerevisiae strains for the productionof hepatitis B virus surface antigen (HBsAg) and European Patentapplication publication No. 0 103 409 relates to the production ofalpha-1-antitrypsin from yeast plasmids.

Protein production in yeast strains indeed presents substantialadvantages over production in bacterial strains. These advantages resultfrom the rather easy growth of yeasts in large scale fermentors and fromthe fact that, contrary to bacteria, yeasts do appear to ressemblemammalian cells in their capacity to add carbohydrate groups to newlysynthesized proteins.

Nevertheless the extraction and purification of the protein from a yeastculture do present technical problems due to the rather complex chemicalcomposition of the yeast cell and more particularly to the presence ofhigh lipid levels when the yeast growth is extended in order to improvethe polypeptide production yield.

For example, the Saccharomyces cell wall is thought to consist of 3layers: (a) an inner layer of alkali-insoluble β-glucan, (b) a middlelayer of alkali-soluble β-glucan and (c) an outer layer of glycoproteinin which the carbohydrate consists of phosphorylated mannan; beneath thecell wall is a cytoplasmic membrane consisting of a very complex mixtureof neutral lipids (mono-, di- and tri-glycerides), free and esterifiedsterols, a complex sphingolipid, glycerophosphatides and neutral as wellas acidic glycolipids; the nucleus contains DNA, various species of RNAand a polyphosphate; vacuoles may contain a great variety of componentsof both high and low molecular weights, they serve as storage vesiclesfor a number of hydrolytic enzymes; the mitochondria are rich in lipid,phopholipid and ergosterol components of the membrane system and thecytoplasm contains a.o. large quantities of ribosomes, polyphosphates,glycogen and a number of glycolytic enzymes. Most yeast cells (such asSaccharomyces species) also contain some amount of lipid in the form ofglobules the amount of which does increase in extended cultures.

A number of multi-steps processes have been disclosed for the extractionand purification of proteins from different sources. Examples referringto proteins produced by engineered microorganisms are those published byTh. STAEHELIN et al. (J. Biol. Chem. 256; 9750-54; 198), K. MURRAY etal. (The EMBO J. 3; p. 645-650; 1984) and R. A. HITZEMAN et al. (Nucl.Ac. Res. 11; 2745-2763; 1983).

Typically, when the produced protein is cell bound, those differentprocesses involve 3 series of steps.

In the first series of steps, the desired protein is removed from thecell interior. Therefore, the cells are either lysed (e.g. by enzymatictreatment) or disrupted (e.g. by mechanical forces (such as shearingforces (e.g. X-press or French press) or shaking with glass beads,eventually with addition of a detergent (see for instance K. MURRAY etal. loc. cit. and R. A. HITZEMAN et al. loc. cit.).

In the second series of steps, the medium is enriched in desiredprotein, e.g. by fractional precipitation by addition of ammoniumsulfate and/or in the presence of polyethylene glycol.

Finally, in the third series of steps, substantially all contaminantsare eliminated from the medium, e.g. by one or several operationsselected from the group comprising ultrafiltration, ion exchange, gelfiltration chromatography and isopycnic gradient centrifugation.

In such process, it is obvious that contaminants like accompanyingproteins (cited by R. A. HITZEMANN et al. loc. cit.), nucleic acids andlipids and more particularly high lipid levels have a harmful influenceon at least one of the steps of the third series (e.g. ultrafiltrationand column chromatography) and the proteolytic enzymes must beeliminated rapidly from the medium. Moreover, it has also been noticedthat ammonium sulfate precipitation is not possible without a previousrough delipidation because lipids interfere with this precipitation.

Therefore it is of prime importance to dispose of a method wherein mostof the contaminants are eliminated before the third series of steps.

Some among the previously described processes disclose the use ofpolyethylene glycol as a selective precipitating agent for proteins anda method for the precipitation of lipoproteins from plasma by usinglipoprotein-polyanion-metal interactions has also been reported.

The method for fractional precipitation of proteins by using nonionicwater-soluble polymers, in particular polyethylene glycol (PEG) has beenintroduced by POLSON et al. (Biochem. Biophys. Acta 82; 463-475; 1964)and discussed by different authors. Among them W. HONIG et al (Analyt.Biochem. 72; 502-512; 1976) mention that "the specificity ofprecipitation, that is the ratio of desired protein and total protein,can be improved by using PEG fractions of lower average molecular weightthan the generally employed PEG 6000". Nevertheless although "bymanipulation of pH concentrates of individual plasma proteins may beobtained" the authors added "however, purification of more complexprotein mixtures such as the supernatant of a cell homogenate isconsiderably poorer".

P. R. FOSTER et al. (Biochim. Biophys. Acta 317; 505-516; 1973 havedescribed a method for the precipitation of enzymes from cell extractsof Saccharomyces cerevisiae by PEG. Methods for the concentration andpurification of viruses and bacteriophages with PEG have been disclosedby B. P. VAJDA (Folia Microbiol. 23, 88-96; 1978) and G. J. LANCZ (Arch.Virusforsch. 42; 303-306; 1973). In the field of hepatitis antigenisolation, the purification of hepatitis B surface antigen (HBsAg) by amethod comprising two successive treatments with PEG 6000 has beendescribed by Ph. ADAMOWICZ et al. (p. 37-49 INSERM SYMPOSIUM No. 18,HEPATITIS B VACCINE, Publ. ELSEVIER, Amsterdam, Holland, 1981). In thismethod of HBsAg purification from plasma, immune complexes and most ofthe lipoproteins are, in a first step, precipitated from serum by PEG6000 at a concentration of 5.5% and, in a second step, HBsAg isprecipitated from the isolated supernatant by addition of PEG at a finalconcentration of 10%.

In the patent literature,

U.S. Pat. No. 3,790,552 discloses a method for removinghepatitis-associated antigen from a protein fraction which comprises astep wherein PEG having a molecular weight 200-6,000 is used in anamount of 12-30% (w/v) for precipitating said antigen.

U.S. Pat. No. 3,951,937 discloses a process for the purification ofhepatitis B antigen (HBAg) involving a double precipitation of HBAg withPEG (4.0-4.5 weight percent) having a molecular weight of at least 600.

U.S. Pat. No. 3,994,870 discloses a method for purifying hepatitis Bantigen (HBAg) wherein HBAg is precipitated by addition of 4.0-4.5weight percent PEG and thereafter subjected to affinity chromatographyutilizing insoluble concavalin A as a chromatographic adsorbent.

European patent application, publication No. 0 112 506 discloses aprocess for producing a hepatitis B infection preventing vaccine fromplasma comprising ammonium sulfate precipitation followed by adsorptionon colloidal silicate and two successive precipitation steps with PEG(having a molecular weight of 2,000-10,000) at a 3-7% (w/v) toprecipitate hepatitis B virus and immune complexes and at a 15-20% (w/v)in the supernatant to precipitate HBsAg.

In the field of alpha-1-antitrypsin isolation, Japanese patentapplication No. 9128-335 (Derwent abstract 84-217127) discloses theprecipitation of alpha-1-antitrypsin from plasma fraction by addition ofPEG in an amount of 15-20% (w/v).

As mentioned above, a method for the precipitation of lipoproteins byusing lipoprotein-polyanion-metal interactions has also been previouslyreported (for instance: M. BURSTEIN et al. Adv. Lip. Res. 11; 67-108;1973 and A. VAN DALEN et al. Biochim. et Biophys. Acta 147; 421-427;1967). This method is performed by interaction between the lipoproteins,a bivalent metal cation and an acidic polysaccharide and, in theseoperative conditions, the amount of precipitate is a function of thebivalent metal cation concentration in the medium.

DESCRIPTION OF THE INVENTION

The starting material for the process of this invention (herein alsoreferred to as `crude extract`) is the supernatant of engineered yeastcells having produced a cell-bound protein and disrupted in the presenceof a non-ionic detergent, as well known in the art.

According to the present invention, there is then made a combination ofthe fractional precipitation by PEG (hereafter refered to as first step)followed by either a fractional precipitation by polyvalent metal cation(more particularly bivalent metal such as calcium and manganese) or byammonium sulfate treatment, said ammonium sulfate treatment beingeventually preceded by ultrafiltration of the supernatant from the firststep.

The present invention derives from the discovery that, when solid PEG(e.g. PEG 6000) is employed at a concentration of 6-12% (w/v) for theextraction of HBsAg from engineered yeast cells disrupted in thepresence of a nonionic detergent, HBsAb does not precipitate. Moreover,by subsequent addition of ammonium sulfate, a two phase system is formedwhich is characterized by the fact that the HBsAg is present in the PEGphase while most contaminants are in the aqueous phase. This result issurprising because, according to the general teaching of the prior artin other crude media, these operative conditions should provoke theprecipitation of the BHsAg. Thus, it is a first object of the presentinvention to use PEG as a solvent for the desired protein produced byyeast cell cultures whereas most of the contaminants are eliminated byprecipitation from the medium.

According to molecular weight, PEG does exist either in solid form or inliquid form, the frontier between both forms being around a molecularweight of 1500.

In the first step of the process according to the invention (which couldalso be considered as a clarification step), PEGs having differentmolecular weights can obviously be used with adequate concentrationadjustment according to the corresponding molecular weight.

For instance, the PEG concentration is preferably 6 to 12% (w/v) whenusing solid PEG (e.g. PEG 6000) and from 10 to 35% (v/v)--and preferablyfrom 20 to 30% (v/v) when liquid PEG (e.g. PEG 300 or 400) is employed.Nevertheless, the use of liquid PEG is preferred for technical reasonsamong which the easiness of later ultrafiltration.

Thus, according to the invention, there is provided a process for theextraction and purification of proteins, more particularly HBsAg oralpha-1-antitrypsin, from the supernatant of yeast cells disrupted inthe presence of a nonionic detergent (preferably a polysorbatedetergent), said supernatant being herein referred to as `crudeextract`, which method comprises a first step wherein either 6-12% (w/v)of solid PEG or preferably 10-25% (v/v) of liquid PEG is added to thecrude extract brought to pH 6 (±0.1), the above limits of PEGconcentrations being mainly fixed by the point at which clarification isreached since further addition of PEG has only detrimental effects, i.e.higher viscosity of the medium and risk of undesired co-precipitation ofHBsAg or alpha-1-antitrypsin.

In the process of the invention, PEG is regarded as a polymerizedorganic solvent favouring a. o. the precipitation of the (lipo)proteinswhose isoelectric point is close to pH 6 and the solubilisation of theother (lipo)proteins, i.e. those which have a markedly differentisoelectric point and those which are highly hydrophobic.

It has been noticed that this PEG clarification step precipitates about75% of the contaminant proteins, 90% of the polysaccharides, 94% of thenucleic acids and 45% of the lipids, while substantially the whole HBsAgor alpha-1-antitrypsin content is recovered in the supernatant.

As indicated hereinabove, the present invention includes a combinationof steps, the first one of them being a clarification step, to yield apartly purified and eventually somewhat opalescent solution of thedesired protein.

In the second step of the process of the invention, the obtained partlypurified solution is either treated with a polyvalent--more particularlybivalent--metal cation such as an aqueous solution of calcium ormanganese chloride at a final concentration of 30 mM or treated withammonium sulfate and this ammonium sulfate treatment is performed eitherafter ultrafiltration of the opalescent solution by addition of solidammonium sulfate up to 40-50% saturation, according to the classicalsalting out method to precipitate the desired protein which can be takenover in a phosphate buffer or by addition of ammonium sulfate up to40-50% saturation to the PEG containing supernatant, forming a two-phasesystem with the desired protein in the PEG phase.

Each of these variations for the second series of steps does provide aclear solution of the desired protein substantially purified regardingits polysaccharides, nucleic acids, lipids and contaminant proteinscontent.

The so-obtained solution can then be processed in the above definedclassical third series of steps, e.g. eventual ultrafiltration, gelfiltration and column chromatography and, in a preferred embodiment ofthe present invention, this third series of steps comprises successivelyan ultrafiltration, a first gel filtration, a column chromatography onweakly alkaline anion exchanger with diethylaminoethyl (DEAE) groups anda second gel filtration to yield a highly purified HBsAg oralpha-1-antitrypsin solution suitable for medical use.

When tested by SDS PAGE polyacrylamide gel electrophoresis, the productrecovered after the second gel permeation is shown to be more than 98%pure with regard to the 23K band which is characteristic of HBsAg ofyeast origin.

Thus, the primary advantage of the method of this invention is that itdoes remove substantially all polysaccharides, nucleic acids, lipids andproteinaceous material and another advantage of the invention is thatthe desired protein may finally be separated in relatively high yield.

When the process of the invention is applied to a crude extract of HBsAgfrom engineered yeast cells, the obtained HBsAg is bound to the nonionic detergent forming therewith composite micelles of a diameter ofabout 22 nm and it has been found that the ratio expressing the amountof non ionic detergent on the amount of proteins, total lipids andpolysorbate in the polysorbate composite micelle is from 15 to 35% (w/v)when the assays are performed by colorimetric method for proteins(LOWRY), total lipids (ZOLLNER) and non ionic detergent (HUDDLESTON andALLRED).

The composite micelles obtained by the process of the invention by usinga polysorbate a non-ionic detergent are novel compounds which are alsoan object of this invention; they are immunogenic and can be formulatedinto vaccine form like classical HBsAg as it is well known in the artfor hepatitis B virus vaccine preparation.

The invention is illustrated by the following examples which are notlimitative of the scope of the invention.

EXAMPLE 1

Pelleted yeast cells (3850 g) of an engineered yeast strain expressionHBsAg which have grown up to 30 g dry cells weight per liter of cultureare suspended in 7.12 liters of Na₂ HPO₄ solution (7.098 g/l).

This suspension is supplemented with 142.5 ml 4% (w/v) EDTA solution,38.5 ml polysorbate 20 and 385 ml isopropanol containing 2.7 gphenylmethylsulfonyl fluoride (PMSF). The pH is adjusted to 8.1 (±0.1)with NaOH (10% w/v in water). The suspension is refrigerated in an icebath and disrupted by 2 passages through a cooled glass beadshomogenizer. The homogenate (crude extract) is then centrifuged for 30minutes at 13000 g.

PEG 400 (2,5 l) is slowly added with stirring to the crude extract (77l) which is maintained below 15° C. The pH is then adjusted to 6 (±0.1)by addition of acetic acid (5M) and the medium is stored for one hour at4° C. before being centrifuged at 7,400-11,000 g for 45 minutes.

The supernatant is brought to pH 7.0 (±0.05) with NNaOH and 300 ml ofMCaCl₂ chloride (i.e. 30 ml per liter of supernatant) is added thereto.The pH is readjusted to 7 (±0.05) with NNaOH and stored overnight at 4°C. The suspension is centrifuged at 3600 g for 45 minutes and thesupernatant is ultrafiltered in an AMICON DC (apparatus sold by AMICONCORP., DANVERS, MA, USA)) equipped with a cartridge having a cut-off of100,000 daltons and thereby first concentrated up to 1/4 (1500 ml) ofits initial volume. The solution is then washed continuously with 12.5 lof 10 mM trometamol adjusted to pH 8 (±0.1) by addition of Nhydrochloric acid (this buffer is hereafter referred to as trometamol pH8) and supplemented with PMSF (1 mM), isopropanol (2.5% v/v) and EDTA (2mM) (final concentrations). The retentate is then further concentratedto 350 ml.

The concentrated solution is applied to a column (φ 10 cm×100 cm)containing 7 l of FRACTOGEL® TSK HW65(F) (a semi-rigid gel consisting ofhydrophilic vinyl polymers with numerous hydroxyl groups on the matrixsurface with particle size 32-62 um manufactured and sold by E. Merck,Darmstadt, FRG) equilibrated in trometamol/HCl buffer 10 mM pH 7 (±0.01)supplemented with 5% (v/v) ethylene glycol.

The HBsAg antigen containing peak is applied to a column (φ 5 cm×30 cm)of 300 ml of FRACTOGEL® TSK DEAE 650 (M) (a weakly basic anion exchangerwherein diethylaminoethyl groups are bound to the hydroxyl groups ofFRACTOGEL TSK HW-65 matrix via ether linkages, manufactured and sold byE. Merck, Darmstadt, FRG) equilibrated in trometamol pH 8 at 4° C. Thecolumn is washed with trometamol pH 8 containing 005MNaCl and HBsAg iseluted with 0.15M NaCl in trometamol pH 8.

The eluate is applied to a column of FRACTOGEL® TSK HW65(F) equilibratedwith Na₂ HPO₄ /NaH₂ PO₄ buffer (10 mM) pH 6.8 supplemented with NaCl(150 mM) yielding a solution of HBsAg/polysorbate composite micelles.

The purification level obtained at each stage of the purification isshown in Table I.

                  TABLE I                                                         ______________________________________                                                      Total    Total Total  Total                                                   HBsAg    Pro-  Polysac-                                                                             Nucleic                                                                              Total                                     Vol    by RIA   teins charides                                                                             acids  lipids                             Fraction                                                                             (1)    (mg)     (g)   (g)    (g)    (g)                                ______________________________________                                        Crude                                                                         extract                                                                              7.7    1232     .259  114    32.5   104                                PEG                                                                           super- 8.02   1100     64    9.5    641    53.7                               natant                                                                        CaCl.sub.2                                                                    super- 8      1190     37    3.8    512    27.2                               natant                                                                        Retentate                                                                            .350   1320     26.7  .744   37.6   3.596                              HBsAg                                                                         peak                                                                          TSK    1.06   736      .935  .014   16     .352                               Eluate                                                                        TSK-   .15    722      .428  .0084  2.6    .275                               DEAE                                                                          HBsAg                                                                         Peak                                                                          TSK    .45    1117     .244  .0094  2.0    .179                               ______________________________________                                         RIA: Radio lmmuno Assay                                                  

The following Table II summarizes the composition of composite micellesof 3 different vaccine batches obtained by the above process withpolysorbate 20.

                  TABLE II                                                        ______________________________________                                                        Total                                                                Proteins Lipids     Polysorbate 20                                                                          Ratio                                           (μg/ml)                                                                             (μg/ml) (μg/ml)                                                                              C                                        Batch  (A)      (B)        (C)       A/B/C                                    ______________________________________                                        I      20       20         8.5       18                                       II     20       16         8.4       19                                       III    20       14.5       15.6      31                                       ______________________________________                                    

EXAMPLE 2

PEG 400 (882 ml) is slowly added to 2,650 l of crude extract prepared asdescribed in example 1 and the pH is adjusted to 6. (±0.1). The mediumis maintained at 4° C. for one hour and then centrifuged at 7400 g. Thesupernatant is concentrated to 1000 ml on an AMICON DC equipped with acartridge having a cut-off of 100,000 daltons and then washed with 3volumes of 20 mM trometamol pH 8. Powdered ammonium sulfate (277 g) isthen added slowly to the retentate, under stirring and at 4° C. After 1hour at 4° C., the precipitate is centrifuged for 15 min. at 1000 g. Thesupernatant is discarded and the precipitate is dissolved in 400 ml 20mM trometamol pH 8 supplemented with 1 mM PMSF. The solution isultrafiltered on an Amicon DC equipped with a cartridge having a cut-offof 10⁶ daltons. The retentate (500 ml) is washed with 2 volumes oftrometamol pH 8 and then applied to a column containing 300 ml ofTSK-DEAE 650M gel equilibrated with trometamol pH 8. When the passage ofthe sample is completed, the column is washed with 0.05M NaCl intrometamol pH 8 and a linear gradient of NaCl (0.05M-0.5M) is thenapplied to the column. The fraction eluted with 0.15M NaCl contains theHBsAg; it is concentrated to 50 ml in an Amicon DC equipped with acartridge having a cut-off of 10,000 and applied to a column (50×100 cm)of Sepharose 4B-Cl (an agarose gel manufactured and sold by PharmaciaFine Chemicals, Uppsala, Sweden) equilibrated with 20 mM trometamol pH 8supplemented with 5% ethylene glycol, yielding a solution ofHBsAg/polysorbate composite micelles.

The purification level obtained at each stage is shown in Table III.

                  TABLE III                                                       ______________________________________                                                          Total    Total Total  Total                                                   HBsAg    Pro-  Polysac-                                                                             Nucleic                                          Vol    by RIA   teins charides                                                                             acids                                 Fraction   (ml)   (mg)     (g)   (g)    (g)                                   ______________________________________                                        Crude extract                                                                            2.650  188      114.721                                                                             36.9   9.250                                 PEG supernatant                                                                          2.530  160      22.755                                                                              3.321  .536                                  Retentate 10.sup.5                                                                       1000   150      7.468 .181   .175                                  AMS-pellet 400    80.8     1.248 .248   .160                                  Retentate 10.sup.6                                                                       1000   72.4     .529  .029   .088                                  Eluate TSK-                                                                              560    44       .157  0.073  .003                                  DEAE                                                                          HBsAg peak                                                                    Sepharose 4B-CL                                                                          200    23       .030  .0003  .00005                                ______________________________________                                         RIA: Radio Immuno Assay                                                  

EXAMPLE 3

HBsAg crude extract (2 liters) is clarified by PEG 400 as described inexample 1. Powdered ammonium sulfate (AMS) (450 g) is added theretounder stirring (final concentration is 45% AMS saturation). Aftersolubilisation of the AMS, the solution is kept at 4° C. for 3 hours atthe end of which period two distinct phases are obtained which areseparated in a separation funnel: a clear (yellow) upper phase (PEG 400phase) and a clear lower phase (aqueous phase), each phase representingabout ±50% of the original volume. The aqueous phase is discarded andthe upper phase is ultrafiltered on a Amicon DC as described for theCaCl₂ supernatant in example 1. The purification procedure is thenfurther carried out as described in example 1 yielding a solution ofHBsAg/polysorbate composite micelles.

The purification level obtained at each stage is shown in Table IV.

                  TABLE IV                                                        ______________________________________                                                          Total    Total Total  Total                                                   HBsAg    Pro-  Polysac-                                                                             Nucleic                                          Vol    by RIA   teins charides                                                                             acids                                 Fraction   (ml)   (mg)     (g)   (g)    (g)                                   ______________________________________                                        Crude extract                                                                            2000   99       55.000                                                                              22.000 5.588                                 PEG supernatant                                                                          2000   92       13.200                                                                              5.680  243                                   PEG phase  1000   98       2.800 1.536  98                                    Retentate  50     98       1.500 .140   6.66                                  HBsAg peak TSK                                                                           123    52       .140  .0077  2.84                                  Eluate TSK-                                                                              25     54       .0377 .0011  .44                                   DEAE                                                                          HBsAg peak TSK                                                                           60     35       .0207 .00154 .39                                   ______________________________________                                         RIA: Radio Immuno Assay                                                  

EXAMPLE 4

HBsAg crude extract (2 liters) is clarified by PEG 400 as described inexample 1. Powdered ammonium sulfate (AMS) (450 g) is added theretounder stirring (final concentration is 45% AMS saturation). Aftersolubilisation of the AMS, the solution is kept at 4° C. for 3 hours atthe end of which period two distinct phases are obtained which areseparated in a separation funnel: a clear (yellow) upper phase (PEG 400phase) and a clear lower phase (aqueous phase), each phase representingabout ±50% of the original volume. The aqueous phase is discarded and aone liter aliquot of PEG phase (upper phase) is diluted twofold with 10mM trometamol pH 8 and applied to a column containing 300 ml ofFRACTOGEL TSK-DEAE 650(M) gel equilibrated with trometamol pH 8 at aflow rate of 100 ml/hour. The gel is washed with trometamol pH 8containing 0.05M NaCl and the HBsAg is then eluted with a NaCl gradient(0.05-0.5 M NaCl in trometamol pH 8). A HBsAg peak is eluted with 0.15MNaCl and, after concentration in an AMICON DC equipped with a cartridgehaving a cut-off of 10,000, the retentate is applied to a column (5×100cm) of Sepharose 4B-Cl equilibrated with trometamol pH 8 containing 5%(v/v) ethylene glycol, yielding a peak of HBsAg/polysorbate compositemicelles.

The purification level at each stage is shown in Table V.

                  TABLE V                                                         ______________________________________                                                          Total    Total Total  Total                                                   HBsAg    Pro-  Polysac-                                                                             Nucleic                                          Vol    by RIA   teins charides                                                                             acids                                 Fraction   (ml)   (mg)     (g)   (g)    (g)                                   ______________________________________                                        Crude extract                                                                            2100   200      60.600                                                                              19.000 4.860                                 PEG phase  1200   210      3.100 1.436  80                                    Eluate TSK-                                                                              460    134      .750  .0048  05                                    DEAE                                                                          HBsAg peak                                                                    Sepharose 4B-CL                                                                          150    110      .120  .0014  0.3                                   ______________________________________                                         RIA: Radio Immuno Assay                                                  

EXAMPLE 5

HBsAg crude extract (500 ml) is clarified by slow addition of a solutionof 50 g PEG 6000 in 160 ml of water under stirring at 4°. The pH isadjusted to 6.1 by addition of 5M acetic acid. After a one hour storageat 4°, the extract is centrifuged for 15 min. at 7000 g. Powderedammonium sulfate (157 g) is then added to the supernatant under stirring(final concentration is 50% in saturated ammonium sulfate). Aftersolubilisation, the solution is kept at 4° C. for 3 hours, after whichperiod two distinct phases are obtained which are separated in aseparation funnel. The upper phase (PEG phase containing the HBsAg) isdiluted twofold with trometamol pH 8 and then applied to a columncontaining 300 ml of TSK-DEAE 650(M) gel equilibrated in trometamol pH 8at a flow rate of 100 ml/hour. The gel is washed with trometamol pH 8containing 0.05M NaCl and the HBsAg antigen is eluted with trometamol pH8 containing 0.15M NaCl, concentrated in an AMICON DC equipped with acartridge having cut-off of 10,000 and then applied to a 2 liters column(5×100 cm) of FRACTOGEL TSK-HW65 (F) equilibrated with trometamol pH 8containing 10% (v/v) ethylene glycol, yielding a solution ofHBsAg/polysorbate composite micelles.

The purification level at each stage is shown in Table VI.

                  TABLE VI                                                        ______________________________________                                                          Total    Total Total  Total                                                   HBsAg    Pro-  Polysac-                                                                             Nucleic                                          Vol    by RIA   teins charides                                                                             acids                                 Fraction   (ml)   (mg)     (g)   (g)    (g)                                   ______________________________________                                        Crude extract                                                                            500    123      22.600                                                                              6.400  1.200                                 PEG supernatant                                                                          490    100      4.153 .509   24                                    PEG phase  270    110      1.820 .2096  85.3                                  Eluate TSK-                                                                              150    66       .258  .028   4                                     DEAE                                                                          HBsAg peak TSK                                                                           60     68       .065  .00048 .084                                  ______________________________________                                         RIA: Radio Immuno Assay                                                  

EXAMPLE 6

PEG 400 (13 ml) is added slowly to alpha-1-antitrypsin crude extract(120 ml) from an engineered yeast culture and the pH is adjusted to 6.1with 5M acetic acid. After a one hour storage at 4° C., the medium iscentrifuged at 5000 g for 15 minutes and 1M CaCl₂ solution is addedslowly to a final concentration of 40 mM. The pH is adjusted to 7 with1M NaOH and, after overnight storage at 4° C., the medium is centrifugedat 7000 g for 15 minutes. The supernatant is diluted twofold with 50 mMtrometamol adjusted to pH 8.7 by addition of 0.1N hydrochloric acid andapplied to a column of FRACTOGEL TSK-DEAE 650(M) (20 ml) equilibratedwith 50 mM trometamol pH 8.7 prepared as indicated above butsupplemented with 5 mM mercaptoethanol. The column is washed withtrometamol pH 8.7 as indicated above but supplemented with 10 mM NaCl, aNaCl gradient is applied (10 mM-250 mM) and the alpha-1-antitrypsin iseluted at a NaCl concentration of 124 mM, separated from a protein peakeluted at 80 mM NaCl. The alpha-1-antitrypsin peak is concentrated in anAMICON DC equipped with a cartridged having a cut-off of 10,000 andapplied to a Sephadex 150 column equilibrated in 50 mM trometamol pH 8.7prepared as indicated above, yielding a solution of purifiedalpha--antitrypsin.

The purification level obtained at each stage is shown in Table VI.

                  TABLE VI                                                        ______________________________________                                                           Total             Total                                                       AAT        Total  Polysac-                                             Vol    by ELISA   Proteins                                                                             charides                                 Fraction    (ml)   (mg)       (g)    (g)                                      ______________________________________                                        Crude extract                                                                             120    180        4.018  .538                                     PEG supernatant                                                                           100    212        1.300  .290                                     CaCl.sub.2 supernatant                                                                    100    194        .660   .110                                     Eluate TSK-DEAE                                                                           520    180        .210   .0052                                    ______________________________________                                         AAT: alpha1-antitrypsin                                                  

EXAMPLE 7

The technique is as described in Example 1 but 300 ml of MMnCl₂ aresubstituted for the 300 ml of MCaCl₂ therein specified. Thecharacteristics of the final product are similar to those of the productobtained at the end of Example 1.

EXAMPLE 8

The technique is as described in Example 1, but 20 ml of Triton X-100 (aproduct manufactures by Rohm and Haas; Darmstadt, FRG) are substitutedfor the 38.5 ml polysorbate 20 therein specified. The characteristics ofthe final product are similar to those of the product obtained at theend of Example 1.

EXAMPLE 9

The technique is as described in Example 1, but 38.5 ml of polysorbate80 are substituted for the 38.5 ml of polysorbate 20 therein specified.The characteristics of the final product are similar to those of theproduct obtained at the end of Example 1.

EXAMPLE 10

The solution of HBsAg composite micelle obtained at the end of Example 1is adjusted to a protein content of 10 ug per milliliter by addition ofNaCl, phosphate buffer (Na₂ HPO₄ /NaH₂ PO₄) and ALHYDROGEL® (analuminium hydroxide gel manufactured and sold by SUPERPHOS Export Co,Copenhagen, Denmark) up to final concentrations of 0.9% (w/v), 20 mM and0.15% (w/v) of Al(OH/₃), respectively, the final pH being 6.9.

The preparation is sterilized and distributed into 2 ml glass vials,each containing one ml dosage unit of vaccine.

EXAMPLE 11

Dosage units of the vaccine of Example 10 have been administered byintramuscular route to two series of seronegative subjects in 2successive injections, at a one month interval. Although theseinjections should be considered as priming administration which shouldbe followed by a booster, e.g. 2 months after the first injection,positive antibody reponses were already noticed one and two monthsrespectively after the first administration.

In the first series (comprising 32 seronegative subjects) one monthafter the first administration, the seroconversion rate was 20/32, i.e.62.5% with a geometric mean titre (GMT) of 9.95 milli internationalunits (MIU) and one month after the second administration theseroconversion rate was 31/32 i.e. 96.9% with GMT 36 MIU.

In the second series (comprising 46 seronegative subjects) one monthafter the first administration the seroconversion rate was 31/46, i.e.67.4% with GMT 13/6 MIU.

Recording of clinical signs and symptoms revealed no temperature riseand no noticeable local reaction among the vaccinees.

What we claim is:
 1. Hepatitis B surface antigen/polysorbate compositemicelle containing from 15 to 35% (w/v) of polysorbate.
 2. Hepatitis Bvaccine containing as active ingredient a composite micelle according toclaim
 1. 3. A method for protecting human against hepatitis B virusinfection comprising administering by intramuscular route to said humana vaccine preparation containing an effective dose of hepatitis Bsurface antigen present in the form of composite micelles according toclaim
 19. 4. The method of claim 3 wherein priming administration isperformed by two injections at one month interval and followed by abooster administration 2 months after the first injection.